Aging is a risk factor for adipose tissue dysfunction, which is associated with inflammatory innate immune mechanisms. Since the adipose tissue/liver axis contributes to hepatosteatosis, we sought to determine age-related adipose tissue dysfunction in the context of the activation of the innate immune system fostering fatty liver phenotypes. Using wildtype and immune-deficient mice, we compared visceral adipose tissue and liver mass as well as hepatic lipid storage in young (ca. 14 weeks) and adult (ca. 30 weeks) mice. Adipocyte size was determined as an indicator of adipocyte function and liver steatosis was quantified by hepatic lipid content. Further, lipid storage was investigated under normal and steatosis-inducing culture conditions in isolated hepatocytes. The physiological age-related increase in body weight was associated with a disproportionate increase in adipose tissue mass in immune-deficient mice, which coincided with higher triglyceride storage in the liver. Lipid storage was similar in isolated hepatocytes from wildtype and immune-deficient mice under normal culture conditions but was significantly higher in immune-deficient than in wildtype hepatocytes under steatosis-inducing culture conditions. Immune-deficient mice also displayed increased inflammatory, adipogenic, and lipogenic markers in serum and adipose tissue. Thus, the age-related increase in body weight coincided with an increase in adipose tissue mass and hepatic steatosis. In association with a (pro-)inflammatory milieu, aging thus promotes hepatosteatosis, especially in immune-deficient mice.

Les Erythropoietin-producing hepatocyte (EPH) sont la plus grande famille de récepteurs tyrosine kinase. Leurs ligands, les éphrines (EFNs), sont aussi des molécules exprimées à la surface cellulaire. Les EPH/EFNs sont impliqués dans de nombreux processus biologiques. L'hypertension artérielle (PA) est une maladie chronique qui, aujourd'hui, est devenue un problème médical critique dans le monde entier et un enjeu de santé publique. La découverte de nouvelles thérapeutiques de l'hypertension sont d'une grande importance pour la santé publique. Jusqu’à tout récemment, il existe seulement quelques études concernant le rôle de l’axe EPH/EFNs sur la fonction des cellules musculaires lisses vasculaires (CMLV). Dans nos études précédentes, nous avons montré qu'EPHB6 et EFNB1, de concert avec les hormones sexuelles, régulent la PA. Dans la présente étude, nous avons constaté que les différents membres de la famille EPH/EFN peuvent réguler soit positivement, soit négativement, la contractilité des CMLV et la PA: tandis que EPHB4 et EFNB2 appartiennent à la première catégorie, EFNB1, EFNB3 et EPHB6 appartiennent à la deuxième. In vivo, des souris males, mais non pas des femelles, porteuses d’une mutation EPHB4 (KO) spécifique du muscle lisse présentent une PA diminuée, comparée aux souris témoins (WT). Les CMLV de souris EPHB4 KO, en présence de testostérone, ont montré une contractilité réduite lors de la stimulation par la phényléphrine (PE). Au niveau moléculaire, la phosphorylation de la protéine kinase II dépendante de Ca2+/calmoduline et de la kinase de la chaine légère de la myosine (CLM) est augmentée, tandis que la phosphorylation de la kinase de la CLM est réduite dans les CMLV KO lors de la stimulation par PE, par rapport au WT CMLV. Cela fournit une base moléculaire à la réduction de la PA et de la contractilité des CMLV chez les souris EPHB4 KO. EFNB2 est le ligand majeur de l’EPHB4. Comme attendu, les souris EFNB2 KO spécifique du muscle lisse avaient un phénotype de PA semblable, quoique non identique, aux souris EPHB4 KO. Les souris mâles EFNB2 KO, mais pas femelles, sous régime régulier ou riche en sel, présentent une PA réduite, par rapport à leurs homologues WT. Au niveau cellulaire, les CMLV des souris KO ont montré une contractilité réduite lors de la stimulation par PE par rapport aux témoins WT. Une région de l’acide aminé (aa) 313 à l’aa 331 dans la partie intracellulaire d’EFNB2 est essentielle pour la signalisation inverse qui régule la contractilité des CMLV, selon des études de mutation-délétion. Dans une étude de génétique humaine, nous avons identifié, dans le gène EFNB2, six SNP qui étaient associées significativement au risque d'hypertension artérielle, de façon dépendante du sexe, ce qui corrobore nos résultats chez les souris. En revanche, la délétion du gène EFNB3 (KO) chez les souris femelles aboutit à une PA élevée et à une augmentation des résistances des petites artères in vivo, améliore la contractilité des petites artères ex-vivo et augmente la contractilité des CMLV in vitro. Les souris mâles KO ont une PA normale, mais la castration conduit à une augmentation significative de la PA dans les souris KO, mais pas dans les souris WT. Les CMLV des souris KO femelles ont montré une phosphorylation accrue de la CLM et une phosphorylation réduite de la kinase de la CLM, ce qui fournit à nouveau une base moléculaire aux phénotypes de PA et de contractilité des CMLV observés. Ce changement de signalisation est attribuable à une protéine adaptatrice Grip1. En effet, dans une étude d'association pan génomique par le Consortium International pour la Pression Sanguine, un SNP dans le gène GRIP1 a approché le seuil de significativité de la valeur p pour son association avec la pression diastolique. Nos recherches, pour la première fois, ont révélé que EPH/EFNs sont de nouveaux composants dans le système de régulation de la PA. Les membres de la famille EPH/EFN peuvent agir comme des forces Yin et Yang pour régler finement le tonus des vaisseaux pour assurer l'homéostasie de la PA et de sa régulation. Ces effets de EPH/EFNs dépendent du sexe et des niveaux d’hormones sexuelles. À partir de ces nouvelles connaissances, nous pourrions développer une nouvelle thérapie personnalisée pour l’hypertension artérielle, utilisant des antagonistes d'hormones sexuelles ou des thérapies de remplacement d'hormones sexuelles, selon les niveaux d'hormones sexuelles des patients et les mutations dans les gènes de l'EPH/EFN. / Erythropoietin-producing hepatocyte (EPH) kinases are the largest family of receptor tyrosine kinases. Their ligands, ephrins (EFNs), are also cell surface molecules. Ephs/EFNs are implicated in many biological processes. Hypertension is a chronic medical condition of high arterial blood pressure (BP). New hypertension therapeutic treatments are of great importance for public health. Until recently, there are only a few studies related to the role of EPHs/EFNs in vascular smooth muscle cell (VSMC) function. In our previous studies, we have found that EPHB6 and EFNB1 function in concert with sex hormones to regulate BP. In the present investigation, we found that different EPH/EFN family members can either positively or negatively regulate the VSMC contractility and BP: while EPHB4 and EFNB2 belong to the former category, EFNB1, EFNB3 and EPHB6, the latter. In vivo, male but not female smooth muscle-specific EPHB4 knockout (KO) mice presented decreased BP, compared to WT controls. VSMCs from EPHB4 KO mice in the presence of testosterone showed reduced contractility. EFNB2 is the major ligand of EPHB4. As expected, smooth muscle-specific EFNB2 KO mice had a similar although not identical BP phenotype as EPHB4 KO mice. Male but not female EFNB2 KO mice on regular or high-salt diet presented reduced BP, compared to WT counterparts. At the cellular level, the KO VSMCs showed reduced contractility compared to WT controls. In a human genetic study, we identified in the EFNB2 gene six SNPs that were significantly associated with hypertension risk in a sex-dependent way, corroborating our findings in mice. On the other hand, EFNB3 gene KO in female mice resulted in elevated BP and small artery resistance in vivo, enhanced small arterial contractility ex vivo, and augmented VSMC contractility in vitro. Male KO mice had normal BP, but castration led to significant BP elevation in KO but not in WT mice. VSMCs from female KO mice showed heightened MLC phosphorylation and reduced MLC kinase phosphorylation. This signaling change was mediated through an adaptor protein Grip1. Indeed, in a genome-wide association study by the International Consortium for Blood Pressure, an SNP in the GRIP1 gene approached the significant threshold p-value for its association with diastolic BP. Our research for the first time revealed that EPHs/EFNs are novel components in the BP regulation system. Members of the EPH/EFN family may act as Yin and Yang forces to finely tune the vessel tone for BP homeostasis and regulation. Such effects of EPHs/EFNs depend on sex and sex-hormone levels. Based on this new knowledge, we could develop novel personalized hypertension therapy using sex hormone antagonists or sex hormone replacement therapy, depending on the sex hormone levels of the patients and mutations in EPH/EFN genes.

La transplantation d’hépatocytes est un procédé séduisant pour remplacer les cellules déficientes dans un foie anatomiquement normal. Dans les maladies métaboliques héréditaires hépatiques (MMHH), la thérapie cellulaire présente un potentiel espoir thérapeutique. Le remplacement d'un pourcentage restreint (5-10%) d’hépatocytes déficients par des hépatocytes normaux pourrait rétablir durablement la fonction métabolique. Les résultats des essais cliniques de transplantation d'hépatocytes génétiquement modifiés ou non sont moins concluants, montrent une prise de greffe insuffisante et, dans la plupart des études, un effet thérapeutique transitoire. L’efficacité limitée de la transplantation d’hépatocytes isolés dans le traitement des MMHH semble en partie liée au faible pourcentage de la masse hépatocytaire reconstituée par les hépatocytes définitivement greffés et fonctionnels. De nombreux modèles animaux ont été développés pour étudier les facteurs pouvant augmenter le nombre et le pourcentage d’hépatocytes transplantés et greffés. Cependant, la majorité de ces modèles ne sont pas transposables en clinique car ils présentent des risques importants ou mal évalués pour les patients. Les principaux objectifs de ce travail ont été d’étudier des moyens peu invasifs pour induire une régénération hépatique et une prise de greffe hépatocytaire significatives dans le but de développer une nouvelle approche de transplantation d’hépatocytes génétiquement modifiés ex vivo pour le traitement de l’hypercholestérolémie familiale. L’effet d’une embolisation portale partielle (EPP) réversible sur la prolifération hépatocytaire et la régénération hépatique a été évalué chez le macaque. A la différence de l’EPP par un produit non résorbable, il ne s’agit pas d’une approche potentiellement délétère à long terme. Une obstruction veineuse plus complète a été provoquée en utilisant le Curaspon®, une gélatine biodégradable, en forme de poudre. Nous avons démontré pour la première fois dans la littérature l’efficacité d’une EPP réversible à induire une importante prolifération hépatocytaire et régénération hépatique. Nos données suggèrent qu’une occlusion portale initiale et temporaire est suffisante pour déclencher les mécanismes responsables d’une régénération hépatique dans le foie non-embolisé. L’utilisation du Curaspon® en poudre peut être considérée comme la forme la plus évoluée d’EPP : très distale, résorbable, qui dure suffisamment pour induire l’hypertrophie hépatique. Cette technique pourrait être indiquée dans des situations cliniques nécessitant une régénération hépatique de courte durée (ex. le traitement des cancers du foie en plusieurs étapes) ou dans des cas qui ne nécessitent pas une résection hépatique, comme la transplantation d’hépatocytes pour le traitement de MMHH. Ces résultats nous ont permis d’évaluer cette approche dans notre protocole préclinique de thérapie génique pour le traitement de l’hypercholestérolémie familiale chez le primate, par autotransplantation d’hépatocytes génétiquement modifiés ex vivo par un vecteur lentiviral. Nous avons démontré que l’EPP réversible induit une régénération hépatique du foie non-embolisé et améliore notablement les résultats de la transplantation d’hépatocytes isolés génétiquement modifiés exprimant la GFP. Seize semaines après la transplantation, les hépatocytes transduits et greffés exprimaient le transgène contrôlé par le promoteur apo-AII humain. Notre protocole a montré pour la première fois chez un gros animal que l’EPP par un produit résorbable entraine avec des conditions de sécurité optimales une repopulation hépatique importante par des hépatocytes transduits par un vecteur lentiviral, et ceci même à distance de la transplantation hépatocytaire. Les résultats encourageants de ces travaux nous ont ouvert la voie pour avancer sur notre projet préclinique et envisager la réalisation d’une étude clinique de phase I/II pour le traitement de l’hypercholestérolémie familiale. / Hepatocyte transplantation is an attractive process for replacing deficient cells in an anatomically normal liver. In metabolic liver diseases, cell therapy could be an interesting alternative to orthotopic liver transplantation. The replacement of a small percentage (5-10%) of deficient hepatocytes by normal hepatocytes could restore the metabolic defect at a long term. Data from clinical studies of hepatocyte autotransplantation or allotransplantation, genetically modified or not, provided poor results, insufficient cell engraftment in the liver parenchyma and, in the majority of cases, a transient therapeutic effect. The limited efficacy of hepatocyte transplantation in metabolic liver diseases is mainly due to the poor percentage of engrafted and finally functional hepatocytes. Numerous animal models have been developed in order to study the factors that could increase the number and the percentage of transplanted and engrafted hepatocytes. However, the majority of these models cannot be used in patients since they present important risks for them. The aim of this work was to evaluate less invasive procedures for inducing liver regeneration and significant hepatocyte engraftment in order to develop a new approach of transplantation of ex vivo genetically modified hepatocytes for the treatment of familial hypercholesterolemia. The effect of reversible portal vein embolization (PVE) on liver regeneration and hepatocyte proleferation was evaluated in monkeys. In contrast to PVE by a permanent embolizing agent, reversible PVE has not a long term deleterious effect on embolized liver. A more complete venous occlusion was obtained by using the powdered form of an absorbable gelatin sponge (Curaspon®). We showed for the first time in the literature the safe and successful use of reversible PVE for inducing significant hepatocyte proliferation and liver regeneration. Our data support that an initial occlusion of the portal branch, even if not permanent, is sufficient to start the mechanisms of liver regeneration in the contralateral lobe. Embolization with Curaspon® powder could be considered to be the ultimate form of embolization: very distal, reversible and lasting sufficiently in order to induce substantial liver hypertrophy. Our findings suggest that this method could reliably be used for clinical purposes, particularly in situations in which short-term regeneration is required (i.e. multi-step management of hepatic malignancies) or in cases where resection of the liver is not finally necessary, such as in hepatocyte transplantation for the treatment of metabolic liver diseases. These promising results on reversible PVE allowed us to evaluate this approach in our preclinical study of gene therapy for the treatment of familial hypercholesterolemia in macaques. Our protocol consisted of an autotransplantation of ex vivo genetically modified hepatocytes by a lentiviral vector. We showed that reversible PVE induces liver regeneration of the non-embolized liver segments and improves considerably hepatocyte transplantation of genetically modified cells expressing Green Fluorescent Protein (GFP). Sixteen weeks after transplantation, transduced engrafted hepatocytes expressed the transgene, which was under control of the human apo-AII promoter. Our protocol showed for the first time in a big animal that PVE by an absorbable agent leads safely to an important and long-term repopulation of the liver by lentivirally transduced hepatocytes. The extremely encouraging results of this work opened our way advancing in our preclinical study and preparing a phase I/II clinical trial for the treatment of familial hypercholesterolemia based on our protocol of autotransplantation of ex vivo genetically modified hepatocytes by a lentiviral vector.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / As liver disease becomes more prevalent, the development of an in vitro culture system to study disease progression and its repair mechanisms is essential. Typically, 2D cultures are used to investigate liver cell (e.g., hepatocyte) function in vitro; however, hepatocytes lose function rapidly when they were isolated from the liver. This has promoted researchers to develop 3D scaffolds to recreate the natural microenvironment of hepatic cells. For example, gelatin-based hydrogels have been increasingly used to promote cell fate processes in 3D. Most gelatin-based systems require the use of physical gelation or non-specific chemical crosslinking. Both of these methods yield gelatin hydrogels with highly interdependent material properties (e.g., bioactivity and matrix stiffness). The purpose of this thesis research was to prepare modularly crosslinked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatic cell fate in 3D. The first objective was to establish tunable gelatin-based thiol-norbornene hydrogels and to demonstrate that the mechanical and biological properties of gelatin hydrogels can be independently adjusted. Furthermore, norbornene and heparin dual-functionalized gelatin (i.e., GelNB-Hep) was prepared and used to sequester and slowly release hepatocyte growth factor (HGF). The second objective was to investigate the viability and functions of hepatocytes encapsulated in gelatin-based hydrogels. Hepatocellular carcinoma cells, Huh7, were used as a model cell type to demonstrate the cytocompatibility of the system. The properties of GelNB hydrogels were modularly tuned to systematically evaluate the effects of matrix properties on cell viability and functions, including CYP3A4 activity and urea secretion. The last objective was to examine the effect of heparin immobilization on hepatocyte viability and functions. The conjugation of heparin onto GelNB led to suppressed Huh7 cell metabolic activity and improved hepatocellular functions. This hybrid hydrogel system should provide a promising 3D cell culture platform for studying cell fate processes.

The studies presented here evaluate the biliary, parenchymal and vascular enhancement effects of two T1-shortening liver-specific contrast agents, Gd-BOPTA and Gd-EOB-DTPA, in Magnetic Resonance Imaging (MRI) of healthy subjects and of patients. Ten healthy volunteers were examined with both contrast agents in a 1.5 T MRI system using three-dimensional gradient echo sequences for dynamic imaging until five hours after injection. The enhancement of the common hepatic duct in contrast to the liver parenchyma was analyzed in the first study. This was followed by a study of the image contrasts of the hepatic artery, portal vein and middle hepatic vein versus the liver parenchyma. While Gd-EOB-DTPA gave an earlier and more prolonged enhancement and image contrast of the bile duct, Gd-BOPTA achieved higher maximal enhancement and higher image contrast for all vessels studied during the arterial and portal venous phases. There was no significant difference in the maximal enhancement obtained in the liver parenchyma. In a third study, another 10 healthy volunteers were examined with the same protocol in another 1.5 T MRI system. Using signal normalization and a more quantitative, pharmacokinetic analysis, the hepatocyte-specific uptake of Gd-EOB-DTPA and Gd-BOPTA was calculated. A significant between-subjects correlation of the uptake estimates was found and the ratio of these uptake rates was of the same magnitude as has been reported in pre-clinical studies. The procedure also enabled quantitative analysis of vascular enhancement properties of these agents. Gd-BOPTA was found to give higher vessel-to-liver contrast than Gd-EOB-DTPA when recommended doses were given. In the final study, retrospectively gathered datasets from patients with hepatobiliary disease were analyzed using the quantitative estimation of hepatic uptake of Gd-EOB-DTPA described in the third study. The uptake rate estimate provided significant predictive ability in separating normal from disturbed hepatobiliary function, which is promising for future evaluations of regional and global liver disease. In conclusion, the differing dynamic enhancement profiles of the liver-specific contrast agents presented here can be beneficial in one context and challenging in another. Diseases of the liver and biliary system may affect the vasculature, parenchyma or biliary excretion, or a combination of these. The clinical context in terms of the relative importance of vascular, hepatic parenchymal and biliary processes should therefore determine the contrast agent for each patient and examination. A quantitative approach to analysis of contrast-enhanced liver MRI examinations is feasible and may prove valuable for their interpretation.

La signalisation calcique joue un rôle crucial dans la réponse des cellules aux signaux extracellulaires. Ces dernières années, les progrès des techniques de microscopie ont permis de montrer que l’organisation spatio-temporelle du signal calcique était un élément fondamental de la réponse des cellules. L'organisation temporelle du signal calcium se caractérise ainsi par l'observation d'oscillations de calcium dont la fréquence varie en fonction du stimulus. Pour de nombreux types cellulaires, et notamment pour les hépatocytes, modèle cellulaire de notre étude, l’amplitude et la fréquence des oscillations de calcium sont très régulières et finement régulées. Les nombreux facteurs qui interviennent pour assurer cette régulation rendent l'étude de ce processus difficile. Ceci explique que l’étude des oscillations de calcium est un des domaines dans lequel l’aspect expérimental est communément complété avec la modélisation mathématique.Parmi les nombreux éléments qui participent à la régulation du signal calcique, les mitochondries qui ont longtemps été considérées comme ayant un rôle passif, pourraient jouer un rôle important. En effet il a été établi que les mitochondries étaient capables d’accumuler de façon active le calcium libéré par le reticulum endoplasmique. Notre objectif a donc été d’étudier l’importance des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes, et grâce à nos résultats expérimentaux, de mettre au point un modèle mathématique. Pour cela nous avons choisi de modifier la prise de calcium par les mitochondries en altérant le moins possible leur fonctionnement normal. Nous avons utilisé une protéine altérant directement la prise de calcium par les mitochondries (HINT 2) et une autre protéine altérant l’interaction entre le reticulum endoplasmique et les mitochondries (R-1). Nos résultats expérimentaux et computationnels démontrent que la protéine HINT 2 augmente l’activité de la chaine respiratoire, ce qui a pour effet d’accélérer l’accumulation de calcium par les mitochondries et d’augmenter la fréquence des oscillations de calcium cytosolique dans les hépatocytes. En effet le modèle élaboré permet à la fois de reproduire les observations expérimentales mais également de prédire avec succès que l’absence de la protéine Hint 2 rend les mitochondries plus sensibles à l’ouverture du pore de transition mitochondrial (mPTP). Nous avons également démontré que le R-1 n’affecte pas significativement la prise de calcium par les mitochondries mais que sa surexpression aboutit à une élévation de la concentration calcique de base des cellules hépatocytaires. A la lumière de nos résultats, cette élévation de la concentration de calcium basale semble être due à son interaction physique avec la pompe ATPase SERCA 2. Nos résultats nous ont permis de mettre en évidence à la fois la contribution des mitochondries dans la régulation des oscillations de calcium dans les hépatocytes grâce à la protéine Hint 2, mais aussi le lien direct entre la surexpression du R-1 et l’élévation de la concentration en calcium de base dans les cellules HepG2. / Calcium signaling plays a crucial role in cell response to external stimuli. These last years, the progress of the microsocopy techniques allowed demonstrate that spatio-temporal organisation of the calcium signal is fundamental for cell physiology. This spatio-temporal organization is characterized by the observation of calcium oscillations whose frequency varies according to the stimulus. For many cell types in particular hepatocytes, calcium oscillations amplitude and frequency are regular and finely regulated. The large range of actors involved in this regulation explains the complexity of studying this process. This explains why the calcium oscillations studies are one of the area in which the experimental approach is commonly associated with computational approaches.Among all the elements participating in the regulation of calcium signaling, mitochondria that have been thought for many years to play a passive role may have a greater role. Indeed it has been shown that mitochondria are able to accumulate actively calcium from the endoplasmic reticulum.Our goal was to study the importance of mitochondria in calcium oscillations regulation in hepatocytes and to elaborate a model based on these experimental results. For that purpose we choose to change the calcium uptake by mitochondria using techniques that didn’t kill them. We used a protein that alters directly mitochondrial calcium uptake (Hint 2), and a protein involved in the interaction between endoplasmic reticulum and mitochondria (R-1).Our experimental and computational results show that HINT 2 enhances the electron transport chain activity, which lead to faster mitochondrial calcium uptake and faster calcium oscillations in the cytosol of hepatocytes. The model allows us not only to reproduce experimental data but also to successfully predict that the loss of HINT 2 lead the mitochondria to be more sensitive to the mitochondrial transition pore opening (mPTP).We also demonstrate that R-1 doesn’t change mitochondrial calcium uptake, but its over expression lead to a higher calcium concentration in resting cells. In the light of our results, this higher calcium concentration seems to be the result of R-1 interaction with ATPase pump SERCA 2.Our results allowed us to show not only the role of mitochondria in the regulation of hepatocytes calcium oscillations by HINT 2, but also the link between R-1 over expression and a higher calcium concentration in the cytosol of HepG2 cells.

Objetivos: Fator de crescimento de hepatócito (HGF) é um polipeptídeo multifuncional, envolvido na embriogênese e regeneração tecidual, com intensa atividade antifibrótica. Os objetivos do presente estudo foram avaliar os efeitos da injeção e reinjeção do HGF coincidindo com seu pico de ação sobre a densidade de colágeno, densidade de vasos, processo inflamatório na lâmina própria e espessura média do epitélio das pregas vocais escarificadas de coelhos. Métodos: Catorze coelhos foram submetidos à injúria em ambas as pregas vocais, subdivididos em grupos 1 e 2. Nos animais do grupo 1 injetou-se HGF nas pregas vocais direitas e nos animais do grupo 2 o HGF foi injetado bilateralmente, sendo reinjetado nas pregas vocais direitas 10 dias após, coincidindo com seu pico de ação. As pregas vocais esquerdas funcionaram como controle, e as laringes foram avaliadas respectivamente aos 30 e 40 dias, através de análise histológica. Resultados: Nossos resultados demonstraram menor densidade de colágeno nas pregas vocais direitas em relação aos controles em ambos os grupos (p=0,018). Densidade de vasos foi maior nas pregas vocais direitas dos animais do grupo 2 (p=0,018); a espessura média do epitélio e o processo inflamatório avaliado na lâmina própria mostraram diferenças estatisticamente não-significantes. Conclusões: A injeção do HGF promoveu menor densidade de colágeno na lâmina própria e a reinjeção levou à menor densidade de colágeno e maior densidade de vasos em pregas vocais escarificadas de coelhos / Objectives: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays various roles in embryogenesis and tissue regeneration and exhibits marked antifibrotic activity. The present study sought to assess the effects of HGF injection and reinjection coinciding with its peak of activity on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in the injured rabbit vocal fold. Methods: Fourteen rabbits were subdivided into two groups and underwent scarring of both vocal folds. Animals in group 1 received HGF injections into the right vocal fold, whereas those in group 2 received bilateral HGF injections and a single reinjection into the right vocal fold 10 days after the first, to coincide with the peak of HGF activity. The left vocal folds served as controls in both groups. Histological assessment of laryngeal specimens was performed at 30 and 40 days respectively. Results: In both groups, collagen density was lower in the right vocal folds than in the left (control) folds (p=0.018). Vessel density was higher in the right vocal folds in group 2 (p=0.018). Differences were found in mean epithelial thickness and inflammatory reaction in the lamina propria, but did not reach statistical significance. Conclusions: In the scarred rabbit vocal fold, HGF injection is associated with decreased collagen density in the lamina propria, whereas reinjection after 10 days produces decreased collagen density and higher vessel density

Objetivos: Fator de crescimento de hepatócito (HGF) é um polipeptídeo multifuncional, envolvido na embriogênese e regeneração tecidual, com intensa atividade antifibrótica. Os objetivos do presente estudo foram avaliar os efeitos da injeção e reinjeção do HGF coincidindo com seu pico de ação sobre a densidade de colágeno, densidade de vasos, processo inflamatório na lâmina própria e espessura média do epitélio das pregas vocais escarificadas de coelhos. Métodos: Catorze coelhos foram submetidos à injúria em ambas as pregas vocais, subdivididos em grupos 1 e 2. Nos animais do grupo 1 injetou-se HGF nas pregas vocais direitas e nos animais do grupo 2 o HGF foi injetado bilateralmente, sendo reinjetado nas pregas vocais direitas 10 dias após, coincidindo com seu pico de ação. As pregas vocais esquerdas funcionaram como controle, e as laringes foram avaliadas respectivamente aos 30 e 40 dias, através de análise histológica. Resultados: Nossos resultados demonstraram menor densidade de colágeno nas pregas vocais direitas em relação aos controles em ambos os grupos (p=0,018). Densidade de vasos foi maior nas pregas vocais direitas dos animais do grupo 2 (p=0,018); a espessura média do epitélio e o processo inflamatório avaliado na lâmina própria mostraram diferenças estatisticamente não-significantes. Conclusões: A injeção do HGF promoveu menor densidade de colágeno na lâmina própria e a reinjeção levou à menor densidade de colágeno e maior densidade de vasos em pregas vocais escarificadas de coelhos / Objectives: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that plays various roles in embryogenesis and tissue regeneration and exhibits marked antifibrotic activity. The present study sought to assess the effects of HGF injection and reinjection coinciding with its peak of activity on collagen density, vessel density, inflammatory reaction in the lamina propria, and mean epithelial thickness in the injured rabbit vocal fold. Methods: Fourteen rabbits were subdivided into two groups and underwent scarring of both vocal folds. Animals in group 1 received HGF injections into the right vocal fold, whereas those in group 2 received bilateral HGF injections and a single reinjection into the right vocal fold 10 days after the first, to coincide with the peak of HGF activity. The left vocal folds served as controls in both groups. Histological assessment of laryngeal specimens was performed at 30 and 40 days respectively. Results: In both groups, collagen density was lower in the right vocal folds than in the left (control) folds (p=0.018). Vessel density was higher in the right vocal folds in group 2 (p=0.018). Differences were found in mean epithelial thickness and inflammatory reaction in the lamina propria, but did not reach statistical significance. Conclusions: In the scarred rabbit vocal fold, HGF injection is associated with decreased collagen density in the lamina propria, whereas reinjection after 10 days produces decreased collagen density and higher vessel density

The chimeric Alb-uPA SCID mouse that has been transplanted with human hepatocytes is a model to facilitate in vivo study of HCV. We explored further development of the model by using repopulation with immortalized human hepatocytes (IHH) in place of primary human hepatocyte (PHH) transplantation to support HCV infection. In vitro HCV studies typically utilize a human hepatoma cell line (Huh7) and rely on transfection with transcribed genomic RNA derived from a unique HCV strain (JFH1). Unfortunately, this system has not been successful in support of infection with serum-derived HCV (HCVser). IHH may offer an alternative since their differentiation status remains close to that of PHH. IHH transfected with HCV RNA (H77 or JFH1) or infected with HCVser showed stable intracellular and supernatant HCV RNA by real-time RT-PCR. IHH showed intracellular HCV NS3 proteins. HCV transfected or infected IHH secrete infectious HCVcc for in vivo and vitro. / Experimental Surgery